Marlene Ricanati

DNA double-strand break rejoining deficiency in TK6 and other human B-lymphoblast cell lines.

TK6, WI-L2, SB and three other B-lymphoblast lines were deficient in the rejoining of DNA double-strand breaks (DSBs) induced by ionizing radiation. Cells of these cell lines rejoin less than 50% of the breaks in 60 min after exposure, as assayed by filter elution at pH 9.6. The deficiency in TK6 cells was confirmed using the comet assay. IN TK6 cells the percentage of DSB rejoining did not vary markedly with dose and was similar for G1, S, and G2 + M-phase cells. Two B-lymphocyte lines (Raji and GM0606), three T-lymphoblast lines (MOLT-4, Jurkat, and CCRF-HSB-2), HL-60 promyelocytes, and GM3440 human skin fibroblasts rejoined more than 50% of the DSBs in this period after exposure. Radiation sensitivity in terms of cell survival was measured in those cells forming colonies. Of the cell lines tested, those that were deficient in DSB rejoining were radiation-sensitive (TK6 and WI-L2: D0 = 0.64 Gy). However, not all lines that were proficient in DSB rejoining were radiation-resistant, since Jurkat and GM0606 cells were relatively radiation-sensitive (D0 = 0.63-0.73 Gy). TK6 and WI-L2 cells were more sensitive to bleomycin (D0 = 8-9 micrograms/ml) than were HL-60 and Raji cells (D0 = 40-54 micrograms/ml). No relationship of DSB rejoining to V(D)J recombinase activity was observed, since no mRNA hybridizing to the cDNA probes for RAG-1 or RAG-2 was detected in any of the cell lines tested.

Relationship between topoisomerase II and radiosensitivity in mouse L5178Y lymphoma strains

The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely related strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. Strain LY-S is sensitive to ionizing radiation relative to strain LY-R and is deficient in the rejoining of DNA double-strand breaks induced by this agent, whereas 2 radiation-resistant variants of strain LY-S have regained the ability to rejoin these double-strand breaks. We have found that the sensitivity of these cells to m-AMSA, VP-16, and ellipticine is correlated to their sensitivity to ionizing radiation. However, this correlation did not extend to their sensitivities to novobiocin, camptothecin, hydrogen peroxide, methyl nitrosourea and UV radiation. Thus, there appears to be a unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors which stabilize the cleavable complex between the enzyme and DNA. It is possible either that (1) topoisomerase II is altered in strain LY-S and that this enzyme is involved in the repair of DNA double-strand breaks or (2) strain LY-S is deficient in a reaction which is necessary for the repair of DNA double-strand breaks induced by ionizing radiation as well as the repair of DNA damage induced by these topoisomerase II inhibitors. m-AMSA, VP-16, and ellipticine were found to be highly mutagenic at the tk locus in L5178Y strains which are heterozygous for the tk gene but not in a tk hemizygous strain, indicating that these inhibitors induce multilocus lesions in DNA, as does ionizing radiation. The differences in the sensitivity of strains LY-R and LY-S to the topoisomerase II inhibitors were paralleled by differences in the induction of protein-associated DNA double-strand breaks in the 2 strains. This correlation did not extend to the radiation-resistant variants of strain LY-S, however. The variants showed resistance to the cytotoxic effects of the inhibitors relative to strain LY-S, but exhibited DNA double-strand break induction similar to that observed in strain LY-S.

Mutagenicity of Photodynamic Therapy as Compared to UVC and Ionizing Radiation in Human and Murine Lymphoblast Cell Lines

Abstract— The mutagenicity of photodynamic therapy (PDT) using red light and either Photofrin® (porfimer sodium) (PF) or aluminum phthalocyanine (AIPc) as the photosensitizer was determined at the thymidine kinase (TK) locus in the human lymphoblastic cell lines, TK6 and WTK1, and was compared to the mutagenicity of UVC and X‐radia‐tion in these cells as well as the mutagenicity of PDT in murine L5178Y lymphoblastic cell lines. Photodynamic therapy was found not to be mutagenic in TK6 cells, which possess an active p53 gene and which are relatively deficient in recombination and repair of DNA double‐strand breaks. In contrast, PDT with either sensitizer was significantly mutagenic in WTK1 cells, which harbor an inactivating mutation in the p53 gene and are relatively efficient in recombination and double‐strand break repair as compared to TK6 cells. The induced mutant frequency in WTK1 cells with PF as the photosensitizer was similar to that induced by UVC radiation but lower than that induced by X‐radiation at equitoxic faiences/ doses. The mutant frequency induced by PDT in WTK1 cells with either photosensitizer was much lower than that induced in murine lymphoblasts at equitoxic fluences. The TK6 and WTK1 cells did not differ in their sensitivity to the cytotoxic effects of PDT, but the level of PDT‐induced apoptosis was greater in TK6 than in WTK1 cells. These results indicate that the mutagenicity of PDT varies in different types of cells and may be related to the repair capabilities as well as the p53 status of the cells.

The Effect of Dose Rate on X-Radiation-Induced Mutant Frequency and the Nature of DNA Lesions in Mouse Lymphoma L5178Y Cells

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.

Induction of Genomic Instability in TK6 Human Lymphoblasts Exposed to 137Cs γ Radiation: Comparison to the Induction by Exposure to Accelerated 56Fe Particles

Abstract Evans, H. H., Horng, M-F., Ricanati, M., Diaz-Insua, M., Jordan, R. and Schwartz, J. L. Induction of Genomic Instability in TK6 Human Lymphoblasts Exposed to 137Cs γ Radiation: Comparison to the Induction by Exposure to Accelerated 56Fe Particles. Radiat. Res. 159, 737–747 (2003). The induction of genomic instability in TK6 human lymphoblasts by exposure to 137Cs γ radiation was investigated by measuring the frequency and characteristics of unstable clones isolated approximately 36 generations after exposure. Clones surviving irradiation and control clones were analyzed for 17 characteristics including chromosomal aberrations, growth defects, alterations in response to a second irradiation, and mutant frequencies at the thymidine kinase and Na+/K+ ATPase loci. Putative unstable clones were defined as those that exhibited a significant alteration in one or more characteristics compared to the controls. The frequency and characteristics of the unstable clones were compared in clones exposed to 137Cs γ rays or 56Fe particles. The majority of the unstable clones isolated after exposure to either γ rays or 56Fe particles exhibited chromosomal instability. Alterations in growth characteristics, radiation response and mutant frequencies occurred much less often than cytogenetic alterations in these unstable clones. The frequency and complexity of the unstable clones were greater after exposure to 56Fe particles than to γ rays. Unstable clones that survived 36 generations after exposure to γ rays exhibited increases in the incidence of dicentric chromosomes but not of chromatid breaks, whereas unstable clones that survived 36 generations after exposure to 56Fe particles exhibited increases in both chromatid and chromosome aberrations.

Diverse Delayed Effects in Human Lymphoblastoid Cells Surviving Exposure to High-LET 56Fe Particles or Low-LET 137Cs Gamma Radiation

Abstract Evans, H. H., Horng, M. F., Ricanati, M., Diaz-Insua, M., Jordan, R. and Schwartz, J. L. Diverse Delayed Effects in Human Lymphoblastoid Cells Surviving Exposure to High-LET 56Fe Particles or Low-LET 137Cs Gamma Radiation. Radiat. Res. 156, 259–271 (2001). To obtain information on the origin of radiation-induced genomic instability, we characterized a total of 166 clones that survived exposure to 56Fe particles or 137Cs γ radiation, isolated approximately 36 generations after exposure, along with their respective control clones. Cytogenetic aberrations, growth alterations, responses to a second irradiation, and mutant frequencies at the Na+/K+ ATPase and thymidine kinase loci were determined. A greater percentage of clones that survived exposure to 56Fe particles exhibited instability (defined as clones showing one or more outlying characteristics) than in the case of those that survived γ irradiation. The phenotypes of the unstable clones that survived exposure to 56Fe particles were also qualitatively different from those of the clones that survived γ irradiation. A greater percentage (20%) of the unstable clones that survived γ irradiation than those that survived exposure to 56Fe particles (4%) showed an altered response to the second irradiation, while an increase in the percentage of clones that had an outlying frequency of ouabain-resistant and thymidine kinase mutants was more evident in the clones exposed to 56Fe particles than in those exposed to γ rays. Growth alterations and increases in dicentric chromosomes were found only in clones with more than one alteration. These results underscore the complex nature of genomic instability and the likelihood that radiation-induced genomic instability arises from different original events.

Induction of multilocus lesions by UVC-radiation in mouse L5178Y lymphoblasts

The survival, the mutant frequency and the nature of the DNA alteration responsible for the inactivation of the thymidine kinase (tk) locus were investigated in 5 strains of mouse L5178Y lymphoblasts exposed to UVC radiation. The nature of the DNA alteration was investigated in independent TK-/- mutants using Southern blot analysis. The concomitant loss of galactokinase (GK) activity in homogenates of individual TK-/- mutants was taken as an indication that the lesion inactivating the tk allele extended to the neighboring galactokinase (gk) allele. The survival of strains LY-R16 and LY-R83 was decreased to a greater extent than that of strains LY-S1, LY-SR1, and LY-3.7.2C, reflecting a deficiency in excision repair in strains derived from LY-R cells. The TK-/- mutant frequency of strain LY-R83, which is monosomic for chromosome 11 and thus hemizygous for the tk and gk genes, was only 50% of the mutant frequency of strain LY-R16 which is heterozygous for the tk gene. Moreover, a greatly reduced percentage of individual spontaneous and UVC-induced TK-/- mutants of strain LY-R83 showed loss of GK activity in comparison to the other strains. This result indicates that UVC irradiation induces intergenic mutations and that such mutants are poorly recovered in the hemizygous strain. Strain LY-3.7.2C appears to have only one active galactokinase (gk) allele, and very few TK-/- mutants of this strain showed loss of GK activity, possibly because this strain, although heterozyogous for the tk gene, is hemizygous in the region of the gk gene. Strains LY-R16 and LY-S1 are deficient in the repair of UVC- and X-radiation-induced damage, respectively, and the percentage of TK-/- mutants with intergenic mutations was higher for strain LY-R16 after UVC-radiation and for strain LY-S1 after X-radiation. These results indicate that unrepaired DNA lesions lead to an increase in intergenic mutations.

Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions

Abstract Evans, H. H., Horng, M-F., Ricanati, M., Diaz-Insua, M., Jordan, R. and Schwartz, J. L. Characteristics of Genomic Instability in Clones of TK6 Human Lymphoblasts Surviving Exposure to 56Fe Ions. Radiat. Res. 158, 687–698 (2002). Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.

Cytotoxic and Mutagenic Effects of Radon and Radon Daughters on Murine L5178Y Lines Differing in DNA Repair

The effects of 222Rn were measured in mouse L5178Y (LY) lymphoblasts that differ in repair capabilities. Line LY-S1 is deficient in the repair of X-radiation-induced DNA doublestrand breaks, while lines LY-R16 and LY-R83 are presumed to be deficient in the excision of UV-radiation-induced pyrimidine dimers. Line LY-R83 is hemizygous while the other two lines are heterozygous at the thymidine kinase (tk) locus. After exposure to radon the D0s were found to be very similar for the three lines (0.30-0.31 Gy), whereas for X radiation the D0 for line LY-S1 is lower (0.7 Gy) than that for the two LY-R lines (1.3 Gy). Mutant frequencies at the tk locus were higher per gray after treatment with radon than X radiation, but at equitoxic doses the mutant frequencies were similar for X and alpha-particle radiation. A low radon-induced mutant frequency was observed for the hemizygous line, in agreement with the hypothesis that multilocus lesions were induced by the alpha-particle radiation and that mutants bearing intergenic lesions were not recovered in the TK+/- line. The entire active tk allele was lost by 81% of the TK-/- mutants of line LY-R16. In lines LY-S1 and LY-R16, 39-43% of the TK-/- mutants exhibited loss of galactokinase activity, indicating that the mutational lesion inactivating the tk gene frequently extended to the neighboring galactokinase gene.

Differential antimutagenicity of WR-1065 added after irradiation in L5178Y cell lines

The purpose of this study was to determine the antimutagenicity of WR-1065 added after irradiation of cells of cell lines differing in their ability to rejoin radiation-induced DNA double-strand breaks (DSBs). The postirradiation antimutagenicity of WR-1065 at the thymidine kinase locus was demonstrated for L5178Y (LY)-S1 cells that are deficient in repair of DNA DSBs. Less postirradiation antimutagenicity of WR-1065 was observed in LY-R16 and LY-SR1 cells, which are relatively efficient in DSB repair. Postirradiation treatment with WR-1065 had only a small stimulatory effect on DSB rejoining. A 3-h incubation of irradiated LY cells with WR-1065 caused slight changes in the distribution of cells in the phases of the cell cycle that differed between LY-S1 and LY-SR1 cells. Both LY-S1 and LY-SR1 cells were protected against the cytotoxic and mutagenic effects of radiation when WR-1065 was present 30 min before and during the irradiation. We conclude that the differential postirradiation effects of WR-1065 in the LY-S1 and LY-SR1 cells are not caused by differences in cellular uptake of the radioprotector or in its radical scavenging activity. Possible mechanisms for the postirradiation antimutagenicity of WR-1065 are discussed.